Process for preparing beta 3 agonists and intermediates

ABSTRACT

The application is directed to efficient and economical processes as described in more detail below for the preparation of the beta 3 agonists of the formula of I-7 and intermediate compounds that can be used for making these agonists. The present disclosure relates to a process for making beta-3 agonists and intermediates using ketoreductase (KRED) biocatalyst enzymes and methods of using the biocatalysts.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase application under 35 U.S.C. §371 of PCT Application No. PCT/US2014/023858, filed Mar. 12, 2014, whichclaims priority under 35 U.S.C. § 119(e) from U.S. ProvisionalApplication Ser. No. 61/791,247, filed on Mar. 15, 2013.

TECHNICAL FIELD

The present disclosure relates to a process for making beta-3 agonistsand intermediates using ketoreductase (KRED) biocatalyst enzymes andmethods of using the biocatalysts.

REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM

The official copy of the Sequence Listing is submitted concurrently withthe specification as an ASCII formatted text file via EFS-Web, with afile name “23146-SeqList.txt”, a creation date of Mar. 7, 2014, and asize of 2.68 kb. The Sequence Listing filed via EFS-Web is part of thespecification and is incorporated in its entirety by reference herein.

BACKGROUND OF THE INVENTION

The application is directed to efficient and economical processes, asdescribed in more detail below, for the preparation of the beta 3agonists of formula I-7 and intermediate compounds that can be used formaking these agonists.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a powder X-ray diffraction pattern of the crystalline saltform of Compound I-5(b) of Example 1.

FIG. 2 is a powder X-ray diffraction pattern of the freebase anhydrousform I of Compound I-7 of Example 1.

SUMMARY OF THE INVENTION

This application is directed to a multiple-step synthetic process formaking a compound of formula I-7 and its intermediates. A KRED enzyme isused in the multiple-step process.

DESCRIPTION OF THE INVENTION

Described herein is a process of making compound I-6, a key intermediatefor making beta-3 agonists, from compound I-3.

In the first embodiment, the multiple-step reactions from compound I-3to compound I-6 comprise the following steps:

(a-2) reducing compound I-3:

in the presence of a KRED enzyme to produce compound I-4:

(b-2) coupling compound I-4 with compound A-1 in the presence ofCatalyst D to produce compound I-5(a)

followed by deprotecting in situ with an acid to produce compound I-5(b)as a salt:

-   -   I-5(a), where R^(N)═P¹ or 1-5(b) where, R^(N)═H;

(c-2) cyclizing and reducing compound I-5(b) in the presence of CatalystE to produce compound I-6 via I-6-1:

-   -   wherein P¹ is selected from the group consisting of Ac, Bn, Boc,        Bz, Cbz, DMPM, FMOC, Ns, Moz, and Ts; and    -   Y is selected from Cl, I, Br, and OTf; and    -   R is selected from the group consisting of H, TMS, TES, TBDMS,        TIPS and TBDPS;    -   and R^(N) is P¹ or H.

Also, described herein is a process of making compound I-7 from compoundI-3 through multiple step reactions:

In the second embodiment, the multiple-step reactions from compound I-3to compound I-7 comprise the following steps:

(a-2) reducing compound I-3:

in the presence of a KRED enzyme to produce compound I-4:

(b-2) coupling compound I-4 with compound A-1 in the presence ofCatalyst D to produce compound I-5(a)

followed by deprotecting in situ with an acid to produce compound I-5(b)as a salt:

-   -   I-5(a), where R^(N)═P¹ or 1-5(b) where, R^(N)═H;

(c-2) cyclizing and reducing compound I-5(b) in the presence of CatalystE to produce compound I-6 via I-6-1:

(d-2) coupling compound I-6 with compound A-2:

in the presence of a coupling agent and optionally including a base toproduce compound I-7;

-   -   wherein P¹ is selected from the group consisting of Ac, Bn, Boc,        Bz, Cbz, DMPM, FMOC, Ns, Moz, and Ts; and    -   X is selected from Na, Li, and K;    -   Y is selected from Cl, I, Br, and OTf; and    -   R is selected from the group consisting of H, TMS, TES, TBDMS,        TIPS and TBDPS;    -   and R^(N) is P¹ or H.

Also described herein is a process of making compound I-6, a keyintermediate for making beta-3 agonists, from compound I-1.

In the third embodiment, the multiple-step reactions from compound I-1to compound I-6 comprise the following steps:

(a-1) reacting compound I-1:

in the presence of a solvent, an oxidizing agent, and Catalyst A to forman aldehyde in situ, followed by a condensation in the presence of X—CNand ammonium salt and a protective reagent, P¹ ₂O or P¹Cl, to producecompound I-2:

(b-1) reacting compound I-2 in the presence of phenyl Grignard reagentto produce compound I-3:

(c-1) reducing compound I-3 in the presence of a KRED enzyme to producecompound I-4:

(d-1) coupling compound I-4 with compound A-1 in the presence ofCatalyst D to produce compound I-5(a)

followed by deprotecting in situ with an acid to produce compound I-5(b)as a salt:

-   -   I-5(a), where R^(N)═P¹ or 1-5(b) where, R^(N)═H;

(e-1) cyclizing and reducing compound I-5(b) in the presence of CatalystE to produce compound I-6 via I-6-1:

-   -   wherein P¹ is selected from the group consisting of Ac, Bn, Boc,        Bz, Cbz, DMPM, FMOC, Ns, Moz, and Ts; and    -   X is selected from Na, Li, and K;    -   Y is selected from Cl, I, Br, and OTf; and    -   R is selected from the group consisting of H, TMS, TES, TBDMS,        TIPS and TBDPS;    -   And R^(N) is P¹ or H.

In one embodiment, the solvent in step (a-1), as set forth in the firstabove embodiment, is selected from the group consisting of THF, MTBE,CH₂Cl₂, MeCN, EtOAc, i-PrOAc, Me-THF, hexane, heptane, DMAc, DMF, methylcyclopentyl ether, toluene and a mixture comprising two or more of theforegoing solvents. In a preferred embodiment the solvent used in step(a-1) is MeCN. In another embodiment, the oxidizing agent is selectedfrom the group consisting of NaOCl, NaClO₂, PhI(OAc)₂, hydrogenperoxide, pyridine sulfur trioxide/Et₃N/DMSO and various Moffattvariants (see Ketones: Dialkyl Ketones. Parkes, Kevin E. B. andRichardson, Stewart K. in Comprehensive Organic Functional GroupTransformations, Volume 3, 1995, Pages 111-204, Editor(s): Katrizky,Alan R.; Meth-Cohn, Otto; Rees, Charles Wayne, Elsevier, Oxford, UK),PCC, DCC, Swern oxidation (oxalic chloride-DMSO-trialkyl amine; seeKetones: Dialkyl Ketones. Parkes, Kevin E. B. and Richardson, Stewart K.in Comprehensive Organic Functional Group Transformations, Volume 3,1995, Pages 111-204, Editor(s): Katrizky, Alan R.; Meth-Cohn, Otto;Rees, Charles Wayne, Elsevier, Oxford, UK) or its variants, TPAP/NMO.

In another embodiment, the oxidation of step (a-1), as set forth in thefirst above embodiment, is carried out using Catalyst A which is TEMPOand its variants including, but not limited to TEMPO/bleach/NaBr,TEMPO/trichloroisocyanuric acid, TEMPO/NCS/TBACl, TEMPO/NCS. In anotherembodiment, Catalyst A is TEMPO or a TEMPO analogue in the presence orabsence of a bromide salt. In another embodiment, the preferred Tempooxidation combination is TEMPO-bleach-bromide salt and TEMPO-PhI(OAc)₂;In a further embodiment a combination of TEMPO-PhI(OAc)₂ with additionaladditives such as HOAc and water is used. In another embodiment, theprotective group is Boc. In a further embodiment, the Boc protectionwith (Boc)₂O is carried out at a temperature of about 35 to about 45° C.using EtOAc or i-PrOAc.

Alternatively, compound I-2 can be prepared via hydrogensulfite adduct.

In one embodiment, the reaction in step (b-1), as set forth in the firstabove embodiment, is carried out at a temperature of about −20° C. toabout 40° C. In another embodiment, the reaction in step (b-1), as setforth in the first above embodiment, is carried out at a temperature ofabout −15° C. to about 5° C.

In another embodiment, the reaction in step (b-1), as set forth in thefirst above embodiment, is carried out in the presence of a solventselected from the group consisting of THF, MTBE, CH₂C₂, Me-THF, hexane,heptane, methyl cyclopentyl ether, toluene and a mixture comprising twoor more of the foregoing solvents.

In another embodiment, the Grignard reagent in step (b-1), as set forthin the first above embodiment, is PhMgBr or PhMgCl.

In one embodiment, the dynamic kinetic reduction in the presence of KREDenzyme in step (c-1 or a2), as set forth in the first, second or thirdembodiments above, is a polypeptide comprising the amino acid sequenceset forth in SEQ ID NO. 1 or an active fragment thereof. In anotherembodiment, the reaction in step (c-1 or a-2), as set forth in thefirst, second or third embodiments above, is carried out in a pH rangeof greater than about pH 8 and higher. In a further embodiment, thereaction in step (c-1 or a-2), as set forth in the first, second orthird embodiments above, is carried out at a pH of about 10±0.5. Inanother embodiment, the reaction of step (c-1 or a-2), as set forth inthe first, second or third embodiments above, is carried out at atemperature range of about 30° C. to about 50° C. In a furtherembodiment, the reaction of step (c-1 or a-2), as set forth in thefirst, second or third embodiments above, is carried out at atemperature range of about 43° C. to about 47° C.

The Sonogoshira coupling reaction carried out in step (d-1 or b-2), asset forth in the first, second or third embodiments above, is thecoupling of a terminal alkyne with an aryl or vinyl halide and isperformed with a palladium catalyst, a copper(I) cocatalyst, or an aminebase (see Sonogoshira, K. In Handbook of Organopalladium Chemistry forOrganic Synthesis; Negishi, E., Ed.; Wiley-Interscience: New York, 2002;pp 493-529.). In one embodiment, Catalyst D used in the Sonogoshirareaction in step (d-1 or b-2) is selected from the group consisting ofPd(PPh₃)₄, PdCl₂, (PPh₃)₂PdCl₂, Pd(dppe)Cl, Pd(dppp)Cl₂, Pd(dppf)Cl₂,and Pd(OAc)₂/Ph₃P or other ligands, in the presence or absence ofcatalytic amount of material selected from CuI, CuBr, and CuCl. In afurther embodiment, the catalyst combination is (PPh₃)₂PdCl₂ and CuI. Inanother embodiment, the reaction in step (d-1 or b-2), as set forth inthe first, second or third embodiments above, is carried out in thepresence of a solvent selected from THF, IPA, MeOH, EtOH, n-PrOH, NMP,DMF, DMAc, MTBE, CH₂Cl₂, MeCN, Me-THF, methyl cyclopentyl ether, andtoluene, and a mixture comprising two or more of the foregoing solvents.In another embodiment, the reaction in step (d-1 or b-2), as set forthin the first, second or third embodiments above, is carried out in thepresence of a solvent made up of a mixture of THF and IPA. In oneembodiment, the acid used in the reaction in step (d-1 or b-2), as setforth in the first, second or third embodiments above, to removalcarbamate protecting group is selected from HCl, HBr, TFA, MeSO₃H,H₂SO₄, p-toluenesulfonic acid, phenylsulfonic acid, camphorsulfonicacid, bromo-camphorsulfonic acid, and other sulfonic acids such asRSO₃H, wherein R is C₁₋₆ alkyl, aryl or substituted aryl. In anotherembodiment, the acid used in the reaction in step (d-1 or b-2), as setforth in the first, second or third embodiments above, is HCl. In afurther embodiment, the reaction product in step (d-1 or b-2), as setforth in the first, second or third embodiments above, is isolated as asolid HCl salt.

In one embodiment, compound I-5(b) acidic salt reacts in step (e-1 orc-2), as set forth in the first, second or third embodiments above, witha base selected from, but not limited to, Et₃N, i-Pr₂NEt, i-Pr₂NH,pyridine, lutidine, N-methyl morphine, t-BuOK, t-BuONa, t-BuOLi, NaH,NaHMDS, LiHMDS, and KHMDS to produce compound I-6-1 through anintramolecular cyclization reaction:

In one embodiment, the base is Et₃N, i-Pr₂NEt, or i-Pr₂NH. In anotherembodiment, the base is i-Pr₂NEt.

In one embodiment, compound I-6-1 is reduced to compound I-6 in thepresence of a catalyst:

The reaction conditions for the conversion from I-6-1 to I-6 can becontrolled using a cis-selective hydrogenation process. In oneembodiment, the cis-selective hydrogenation of step (e-1 or c-2), as setforth in the first, second or third embodiments above, is carried out inthe presence of Catalyst E selected from the group consisting of Pt onalumina, Pd on alumina, Rh on alumina, Pd/C, Pd(OH)₂—C, Pt on alumina-Von carbon or vanadate, Raney Ni, Rh/C, Rh/Al, Pt/C, Ru/C and PtO₂. Inanother embodiment, Catalyst E is Pt on alumina.

In another embodiment, the cis-selective hydrogenation from I-6-1 to I-6in step (e-1 or c-2) is carried out in the presence of a hydroxylprotecting reagent, which protects the hydroxy group in situ andtherefore improves the diastereoselectivity. Protecting reagents can beselected from the TMSCl, HMDS, TESCl, TIPSCl, and TBDMSCl. In anotherembodiment, the protecting reagent is TMSCl.

In one embodiment, the hydrogenation reaction of step (e-1 or c-2), asset forth in the first, second or third embodiments above, is carriedout at about 10° C. to about 70° C. In another embodiment, thehydrogenation reaction of step (e-1 or c-2), as set forth in the first,second or third embodiments above, is carried out at about 20° C. toabout 50° C. In a further embodiment, the hydrogenation reaction of step(e-1 or c-2), as set forth in the first, second or third embodimentsabove, is carried out stagewise at about 20° C. and then at about 50° C.

In another embodiment, the reduction reaction of step (e-1 or c-2), asset forth in the first, second or third embodiments above, is carriedout in the presence of hydrogen gas. In another embodiment, the pressureof the hydrogen gas ranges from about 15 to about 400 psi. In a furtherembodiment, the pressure of the hydrogen gas ranges from about 50 toabout 100 psi.

The reaction between I-6 and A-2 can be carried out in the presence of acoupling reagent. Suitable coupling reagents include, but are notlimited to, CDI, DCC, EDC, EDC methiodide, T3P, HATU, HBTU andmix-anhydrides. In another embodiment, the coupling agent is DCC, EDC,or EDC methiodide. In a further embodiment, the coupling reagent is EDC.

The reaction between I-6 and A-2 can be carried out in the presence of asolvent while the substrate is treated with an acid such as HCl, MeSO₃H,H₂SO₄ to selectively protect the secondary pyrrolidine amine. Suitablesolvents include, but are not limited to, both aqueous and non-aqueoussolvents such as MeOH, EtOH, isopropyl alcohol (IPA), n-PrOH, MeCN, DMF,DMAc, NMP, THF, EtOAc, IPAc, or toluene.

A promoter can be used in the reaction between I-6 and A-2. Suitablepromoters include, but are not limited to, HOBT and HOPO.

Suitable pH values for the reaction between I-6 and A-2 can be about 2.5to about 5.0, or more specifically, about 3.0 to about 4.0, or even morespecifically, about 3.0 to about 3.5. The pH can be adjusted to thedesired ranges using an acid such as HCl, HBr, HI, HNO₃, H₂SO₄, H₃PO₄,TFA and MeSO₃H. In one embodiment, the pH is about 3.0 to about 3.7. Inanother embodiment, the pH is about 3.3 to about 3.5.

In one embodiment, the reaction in step (d-2), as set forth in thesecond or third embodiments above, is carried out in the presence of abase. In a further embodiment, the reaction in step (d-2), as set forthin the second or third embodiments above, is carried out in the presencepyridine or pyridine salt. Pyridine salt includes, but not limited tothe corresponding HCl salt, H₂SO₄ salt, H₃PO₄ salt, HBr salt, HI salt,HNO₃ salt, or MeSO₃H salt.

In the first, second or third embodiments listed above, the KRED enzymecomprises a polypeptide sequence set forth in SEQ ID NO.1 or an activefragment thereof.

In the first, second or third embodiments listed above, a cofactorrecycling system is also present in addition to a KRED enzyme. Suitablecofactor recycling systems include, but are not limited to, a KREDenzyme such as polypeptide of SEQ ID NO.1 and a glucose deydrogenaseenzyme.

In the first, second or third embodiments listed above, a KRED enzymecomprising a polypeptide sequence set forth in SEQ ID NO.1 or an activefragment thereof and a cofactor recycling system are present in thereduction from I-3 to I-4.

In the first, second or third embodiments listed above, a cofactormolecule which can donate a hydride is also present in addition to aKRED enzyme. In one embodiment, the cofactor is selected from the groupconsisting of NADH and NADPH.

In the first, second or third embodiments listed above, a co-substratemolecule which can provide a hydride for the recycling of a cofactormolecule is also present in addition to a KRED enzyme. In oneembodiment, the co-substrate is selected from the group of secondaryalcohols including but not limited to 2-propanol, 2-butanol, 2-pentanol,2-hexanol, 2-heptanol, and 2-octanol.

In the first, second or third embodiments listed above, the enzymaticreduction of I-3 to I-4 is carried out in a solvent. Suitable solventscan be selected from the group consisting of 2-propanol, sec-butanol,iso-butanol, 2-pentanol, 2-hexanol, 2-heptanol, 2-octanol, DMSO, DMF,DMAc, and NMP, and combinations thereof. In one embodiment, the solventis 2-propanol.

In the first, second or third embodiments listed above, a suitabletemperature for the dynamic kinetic resolution (DKR) reduction from I-3to I-4 ranges from about 0° C. to about 60° C., or more specifically,from about 30° C. to about 50° C., or even more specifically, from about43° C. to about 47° C. In one embodiment, the temperature is about 45°C.

In the first, second or third embodiments listed above, a KRED enzyme iscoupled with a cofactor recycling system and an NADPH cofactor is usedto reduce compound I-3 to obtain compound I-4. Suitable reactionconditions for the KRED-catalyzed reduction of I-3 to I-4 are providedbelow and in the Examples.

In the first, second or third embodiments listed above, the KRED enzymeor active fragment thereof can be immobilized on a solid support. Insome embodiments, the KRED enzyme or active fragment thereof havingketoreductase activity of the present disclosure can be immobilized on asolid support such that they retain their improved activity,stereoselectivity, and/or other improved properties relative to thereference polypeptide of SEQ ID NO: 1 or active fragment thereof. Insuch embodiments, the immobilized polypeptides can facilitate thebiocatalytic conversion of the substrate of compound I-3 or structuralanalogs thereof to the product of compound I-4 or correspondingstructural analogs (e.g., as shown in the process of Scheme 1 describedherein), and after the reaction is complete are easily retained (e.g.,by retaining beads on which polypeptide is immobilized) and then reusedor recycled in subsequent reactions. Such immobilized enzyme processesallow for further efficiency and cost reduction. Accordingly, it isfurther contemplated that any of the methods of using the KRED enzyme oractive fragment thereof of the present disclosure can be carried outusing the same KRED enzyme or active fragment thereof bound orimmobilized on a solid support.

Methods of enzyme immobilization are well-known in the art. The KREDenzyme can be bound non-covalently or covalently. Various methods forconjugation and immobilization of enzymes to solid supports (e.g.,resins, membranes, beads, glass, etc.) are well known in the art anddescribed in e.g.: Yi et al., “Covalent immobilization of ω-transaminasefrom Vibrio fluvialis JS17 on chitosan beads,” Process Biochemistry42(5): 895-898 (May 2007); Martin et al., “Characterization of free andimmobilized (S)-aminotransferase for acetophenone production,” AppliedMicrobiology and Biotechnology 76(4): 843-851 (September 2007);Koszelewski et al., “Immobilization of ω-transaminases by encapsulationin a sol-gel/celite matrix,” Journal of Molecular Catalysis B:Enzymatic, 63: 39-44 (April 2010); Truppo et al., “Development of anImproved Immobilized CAL-B for the Enzymatic Resolution of a KeyIntermediate to Odanacatib,” Organic Process Research & Development,published online: dx.doi.org/10.1021/op200157c; Hermanson, G. T.,Bioconjugate Techniques, Second Edition, Academic Press (2008); Mateo etal., “Epoxy sepabeads: a novel epoxy support for stabilization ofindustrial enzymes via very intense multipoint covalent attachment,”Biotechnology Progress 18(3):629-34 (2002); and BioconjugationProtocols: Strategies and Methods, In Methods in Molecular Biology, C.M. Niemeyer ed., Humana Press (2004); the disclosures of each which areincorporated by reference herein.

Solid supports useful for immobilizing the KRED enzyme of the presentdisclosure include but are not limited to beads or resins comprisingpolymethacrylate with epoxide functional groups, polymethacrylate withamino epoxide functional groups, styrene/DVB copolymer orpolymethacrylate with octadecyl functional groups. Exemplary solidsupports useful for immobilizing the KRED enzyme of the presentdisclosure include, but are not limited to, chitosan beads, Eupergit® C,and SEPABEADS® (Mitsubishi Chemical Company), including the followingdifferent types of SEPABEAD®: HP2MG; EC-EP, EC-HFA/S; EXA252, EXE119 andEXE120. In some embodiments, the solid support can be a bead or resincomprising carbonate.

KRED enzymes belonging to class of oxidoreductases are useful for thesynthesis of optically active alcohols from the correspondingpro-stereoisomeric ketone substrates by stereospecific reduction ofcorresponding racemic aldehyde and ketone substrates. Isolatedketoreductases require the presence of a nicotinamide cofactor. Hydrogenand two electrons are transferred from the reduced nicotinamide cofactor(NADH or NADPH) to the carbonyl group of the substrate to effect areduction to the chiral alcohol.

In the first, second or third embodiments listed above, compound I-7 isobtained in the form of a crystalline anhydrous free base. In the first,second or third embodiments listed above, compound I-7 is obtained inthe form of a crystalline free base hemihydrate.

As used herein, the term “alkyl” means both branched- and straight-chainsaturated aliphatic hydrocarbon groups having the specified number ofcarbon atoms. For example, C₁₋₆ alkyl includes, but is not limited to,methyl (Me), ethyl (Et), n-propyl (Pr), n-butyl (Bu), n-pentyl, n-hexyl,and the isomers thereof such as isopropyl (i-Pr), isobutyl (i-Bu),secbutyl (s-Bu), tert-butyl (t-Bu), isopentyl, sec-pentyl, tert-pentyland isohexyl.

As used herein, the term “aryl” refers to an aromatic carbocycle. Forexample, aryl includes, but is not limited to, phenyl and naphthalyl.

Throughout the application, the following terms have the indicatedmeanings unless noted otherwise:

Term Meaning Ac Acyl (CH₃C(O)—) Bn Benzyl BOC (Boc) t-Butyloxycarbonyl(Boc)₂O Di-tert-butyl dicarbonate t-BuOK Potassium tert-butoxide t-BuOLiLithium tert-butoxide t-BuONa Sodium tert-butoxide Bz Benzoyl CbzCarbobenzyloxy CDI 1,1′Carbonyldiimidazole DCCN,N′-Dicyclohexycarbodiimide DCM Dichloromethane DKR Dynamic kineticresolution DMAc N,N-dimethylacetamide DMAP 4-Dimethylaminopyridine DMFN,N-dimethylformamide DMPM 3,4-Dimethoxybenzyl DMSO Dimethyl sulfoxideDABCO 1,4-diazabicyclo[2.2.2]octane dr (Dr) diastereomer ratio EDC1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide ee enantiomeric excess EtEthyl EtOAc Ethyl acetate FMOC 9-Fluorenylmethyloxycarbonyl HATUO-(7-Azabenzotriazol-1-yl)-N,N,N′,N′- tetramethyluroniumhexafluorophosphate HBTU O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate) HMDS Hexamethyldisilazane HOBT1-Hydroxy-1H-benzotriazole HOPO 2-Hydroxypyridine-N-oxide Immobead ™150A an apolar enzyme carrier bead of macroporous acrylic polymer IPAIsopropyl Alcohol KHMDS Potassium bis(trimethylsilyl)amide KREDKetoreductase LC/MS or LC-MASS Liquid chromatography mass spectrum LCAPLiquid Chromatography Area Percent Me Methyl Moz or MeOZ p-Methoxybenzylcarbonyl MTBE Methyl tert-butyl ether NaHMDS Sodium hexamethyldisilazideNADP Nicotinamide adenine dinucleotide phosphate sodium salt NMPN-Methyl-2-pyrrolidone Ns 4-Nitrobenzene sulfonyl OTf Triflate PCCPyridinium chlorochromate 5% Pd/Al₂O₃ Palladium, 5 weight percent onaluminum oxide 5% Pd/C Palladium, 5 weight percent on activated carbon10% Pd/C Palladium, 10 weight percent on activated carbon PdCl₂Palladium (II) chloride (PPh₃)₂PdCl₂Bis(triphenylphosphine)palladium(II) dichloride Pd(dppe)Cl1,2-bis(diphenylphosphino)ethane chloropalladium Pd(dppp)Cl₂ 1,3-bis(diphenylphosphino)propane dichloropalladium(II) Pd(dppf)Cl₂1,1′-bis(diphenylphosphino)ferrocene dichloropalladium(II) PhI(OAc)₂Iodobenzene diacetate PhMgBr Phenyl Magnesium Bromide (PPh₃)₂PdCl₂Triphenyl phospate Paladium Chloride 5% Pt/Al₂O₃ Platinum, 5 weightpercent on aluminum oxide Ph Phenyl i-PrOAc Isopropyl acetate PrOHPropyl alcohol i-Pr₂NEt Diisopropylethylamine (DIPEA) i-Pr₂NHDiisopropylamine 5% Rh/Al₂O₃ Rhodium, 5 weight percent on aluminum oxideT3P Propane Phosphonic Acid Anhydride TBACl Tetrabutylammonium chlorideTBDMS t-Butyldimethylsilyl TBDPS tert-Butyldiphenylsilyl ether TBDMSClTert-Butyldimethylsilyl chloride TEA or Et₃N Triethylamine TEMPO1-Oxyl-2,2,6,6-tetramethylpiperidine TES Triethylsilyl TESClTriethylchlorosilane TFA Trifluoroacetic acid THF Tetrahydrofuran TIPSTriisopropylsiyl TIPSCl Triisopropylchlorosilane TMS TrimethylsilulTMSCl Trimethylchlorosilane TPAP/NMO Tetrapropylammonium Perruthenate/N-Methylmorpholine-N-Oxide Ts p-Toluene sulfonyl XRPD x-ray powderdiffraction

Reaction Schemes below illustrate the synthetic steps, reagents andconditions employed in the synthesis of the compounds described herein.The synthesis of compound I-7 which is the subject of this invention maybe accomplished by one or more of similar routes.

EXAMPLE 1

Preparation of Compounds I-6 and I-7

-   -   Compounds I-6 and I-7 can be made from compound I-1 as described        below in Scheme 1. NOTE: Compounds appearing within brackets [ ]        denote non-isolated intermediates.

In Scheme 1, starting from 4-pentyn-1-ol (I-1), I-2 was prepared withoutisolating any intermediates via a TEMPO-PhI(OAc)₂ oxidation followed bya Strecker reaction (see Nitriles: General Methods and AliphaticNitriles. North, Michael. in Comprehensive Organic Functional GroupTransformations, Volume 3, 1995, Pages 611-640, Editor(s): Katrizky,Alan R.; Meth-Cohn, Otto; Rees, Charles Wayne, Elsevier, Oxford, UK) andin situ Boc protection. Compound I-2 could be isolated as a solid.Treatment of I-2 with phenyl Grignard gave a racemic ketone I-3, whichwas then selectively transformed to optically pure alcohol I-4 underenzymatic (dynamic kinetic resolution) DKR reduction conditions, as thetwo stereogenic centers were set up in one step. I-5 was then preparedvia a Sonogishira reaction followed by deprotection and isolated as acrystalline HCl salt. Treatment of I-5HCl salt with Hunig's base(Diisopropylethylamine; see Ketones: α,β-Unsaturated Ketones. Ebenezer,Warren J. and Wight, Paul. in Comprehensive Organic Functional GroupTransformations, Volume 3, 1995, Pages 205-276, Editor(s): Katrizky,Alan R.; Meth-Cohn, Otto; Rees, Charles Wayne, Elsevier, Oxford, UK). Inwarm DMAc-THF gave cyclized intermediate I-6-1, which was furthertreated with TMSCl to protect the OH in situ. As such, a subsequentone-pot stereoselectively hydrogenated afforded the desired cis isomerin high yield. Finally, EDC coupling of I-6 and sodium salt ofpyrimidinone acid A-2 completed the preparation of I-7. Detailedexperimental conditions are described below.

Step 1. Preparation of Compound I-2 from Compound I-1

A 250 mL 3-neck round bottom flask equipped with an overhead stirrer wascharged with 4-pentyn-1-ol (I-1) (10 g, 0.119 mol), acetonitrile (100mL), water (0.5 mL), acetic acid (1.0 mL) and TEMPO (0.92 g, 5.8 mmol).Iodobenzene diacetate (39.4 g, 0.124 mol) was charged in portions over2.5 hours, while the internal temperature was maintained at 20-25° C.with a cold water bath. The reaction was aged 3.5 hours at roomtemperature. The solution was used directly in subsequent Streckerreaction.

A 500 mL 3-neck round bottom flask equipped with an overhead stirrer wascharged with water (40 mL), ammonium hydroxide (28 wt %, 40 mL),ammonium acetate (11.45 g, 0.149 mol) and potassium cyanide (9.68 g,0.149 mol). After stirring to dissolve solids, the above crude aldehydesolution from the oxidation was added drop-wise while the internaltemperature was maintained <40° C. The reaction was agitated at 45° C.for 7-10 hours. Acetonitrile was removed under reduced pressure whilethe internal temperature was maintained <25° C. EtOAc (200 mL) wasadded. The organic phase was separated.

Step 2. Crystallization of Compound I-2

To above crude organic solution was charged (BOC)₂O (26.0 g, 0.119 mol).The reaction mixture was gradually warmed to 35-40° C. and aged at35-40° C. for additional 14 hours. The batch was cooled to ambienttemperature and an aqueous solution of 7.5 wt % NaHCO₃ (50 mL). Theseparated organic layer was solvent switched to heptane at final volumeof ˜300 mL in vacuum, while the internal temperature was maintained at35-45° C. The resulting slurry was aged 2 hours at 20° C. beforefiltration. The wet cake was washed with heptane (100 mL), and vacuumoven-dried at 40° C. to give 18.3 g of the product compound I-2. ¹H NMR(400 MHz, CDCl₃): δ 5.09 (s, br, 1H), 4.74 (s, br, 1H), 2.43 (m, 2H),2.08 (t, J=2.8 Hz, 1H), 2.04 (m, 2H), 1.47 (s, 9H).

¹³C NMR (100 MHz, CDCl₃): δ 154.4, 118.5, 81.4, 70.9, 41.8, 32.1, 28.4,15.1.

Alternative Crystallization Procedure for Compound I-2

The separated organic phase was concentrated to ˜50 mL in vacuum, whichwas then added to a slurry of heptane (375 mL) seeded with product (125mg) over several hours at 15-25° C. The batch was then solvent switchedto heptane in vacuum at final volume of 250 mL, maintaining the internaltemperature below 30° C. The resulting slurry was aged 2 hours at 20° C.before filtration. The wet cake was washed with heptane (100 mL), andvacuum oven-dried at 40° C. to give 18.3 g of the product Compound I-2.

Step 3. Preparation of Compound I-3 from Compound I-2

To a solution of PhMgBr (1Molar, “M”, in THF, 106 mL, 0.106 mol) at −10°C. was added a solution of Boc aminonitrile I-2 (10 g, 0.048 mol) in THF(20 mL) dropwise over 2-3 hours. The reaction slurry was aged at 0° C.for additional 4 hours. The reaction mixture was then added to anaqueous solution of 10 wt % citric acid (135 mL) at −5° C. to 0° C. over0.5 to 1 hour, maintaining the internal temperature below 20° C. Thequenched biphasic solution was agitated at ambient temperature for 1 hand the layers were separated. The organic phase was washed with 10%brine (20 mL). The organic was azeotropically solvent-switched toisopropanol at a final volume of ˜65 mL. The solution was heated to 35°C. and water (˜25 mL) was added. The batch was seeded. After aging 30minutes at 35° C., water (65 mL) was added dropwise over 1 hour. Theslurry was aged an additional 30 minutes at 35° C., then, cooled toambient temperature. The batch was agitated several hours beforefiltration. The wet cake was washed with 30% isopropanol in water (40mL). Vacuum oven dry at 50° C. with nitrogen sweep gave Compound I-3. ¹HNMR (400 MHz, CDCl₃): δ 8.02 (m, 2H), 7.61 (m, 1H), 7.50 (m, 2H), 5.47(m, 1H), 5.42 (m, 1H), 2.37 (m, 1H), 2.27 (m, 1H), 2.15 (m, 1H), 2.01(t, J=2.3 Hz, 1H), 1.75 (m, 1H), 1.46 (s, 9H).

¹³C NMR (100 MHz, CDCl₃): δ 198.9, 155.8, 134.6, 134.1, 129.1, 129.0,83.3, 80.2, 69.6, 54.5, 32.9, 28.6, 15.2.

Step 4. Preparation of Compound I-5 from Compound I-3

To a solution of sodium tetraborate decahydrate (18.7 g, 4.9 mmol) inwater (1 L) at ambient temperature was added 5 Normal (“N”) NaOH to pHof approximately 10. DKR enzyme (2 g) with the SEQ. ID. NO. 1 was addedand dissolved with gentle agitation. A solution of beta-NADP-Na (0.2 g,0.26 mmol) in water (20 mL) followed by a solution of phenyl ketone I-3(100 g, 0.348 mol) in i-PrOH (1 L) was added at ambient temperature withgood mixing. The batch was then agitated at 45° C. for at least 24hours. The batch was cooled to ambient temperature and MTBE (1 L) wasadded. The separated aqueous phase was extracted with MTBE (0.5 L) andi-PrOH (0.5 L). The combined organic phase was washed with water (1 L).The separated organic phase was then azeotropically dried in vacuum at afinal volume of ˜0.3 L, maintaining the internal temperature below 45°C. The crude solution was directly used for next step. ¹H NMR (400 MHz,CDCl₃): major rotomer: δ 7.34 (m, 4H), 7.28 (m, 1H), 4.75 (s, br, 1H),4.73 (m, 1H), 3.82 (m, 1H), 3.27 (s, 1H), 2.27 (m, 2H), 1.97 (t, J=2.6Hz, 1H), 1.83 (m, 1H), 1.72 (m, 1H), 1.37 (3, 9H). ¹³C NMR (100 MHz,d₆-DMSO): major rotomer: δ 156.7, 141.9, 128.5, 127.9, 126.5, 83.9,79.9, 76.0, 69.1, 56.2, 30.6, 28.5, 15.7.

To crude solution was added THF (0.3 L) followed by p-iodonitrobenzene(90.4 g, 0.363 mol). After vacuum degass with nitrogen,bisotriphenylphosphine palladium (II) dichloride (2.43 g, 0.35 mmol)followed by CuI (1.3 g, 0.68 mmol) and triethyl amine (36.7 g, 0.363mol) was added. The batch was agitated at ambient temperature for 3 h.i-PrOAc (1 L) was added. The batch was washed with 10% aq NH₄Cl (2×0.3L) twice, 1 N HCl (0.3 L), and water (0.3 L). The separated organicphase was azeotropically solvent-switched to i-PrOH in vacuum to give aslurry at a final volume of ˜0.6 L, maintaining the internal temperaturebelow 45° C. Concentrated HCl (37%) was added and the batch was thenagitated at 60° C. for 5 hours. The slurry was cooled to ambienttemperature and aged for 3 hours before filtration. The wet cake wasdisplacement washed with i-PrOH (0.2 L) followed by THF (2×0.2 L). Thewet cake was then slurried in THF (0.76 L) at 60° C. for 4 hours. Theslurry was filtered and displacement washed with THF (2×0.2 L). Vacuumoven drying at 40° C. with nitrogen sweep gave Compound I-5HCl salt. ¹HNMR (500 MHz, CD₃OD): δ 8.19 (m, 2H), 7.55 (m, 2H), 7.47 (m, 2H), 7.41(m, 2H), 7.36 (m, 1H), 4.71 (d, J=7.8 Hz, 1H), 2.54 (t, J=7.2 Hz, 2H),1.88 (m, 2H).

¹³C NMR (125 MHz, CD₃OD): δ 148.6, 142.0, 133.7, 131.6, 130.1, 129.9,128.2, 124.7, 94.4, 81.5, 74.3, 57.9, 29.7, 16.8.

The crystalline HCl salt form of Compound I-5(b) can be characterized byXRPD by the following reflections with the d-spacing.

Position [°2 Theta] d-spacing [Å] 4.2174 20.95196 8.5827 10.3028 12.92826.84784 13.5177 6.55051 16.65 5.32461 20.9425 4.24193 22.6974 3.9177724.363 3.65356 24.6986 3.60468 28.1911 3.16555Step 5. Preparation of Compound I-6 from Compound I-5(b)

To a slurry of amino alcohol HCl salt (I-5(b)) (100 g, 0.288 mol) in THF(500 mL) and DMAc (100 mL) was added i-Pr₂NEt (151 mL, 0.87 mol)dropwise under nitrogen, while the internal temperature was maintainedbelow 25° C. The batch was agitated at 55-65° C. for 3-5 hours, thencooled to 0-15° C. TMSCl (55.3 mL, 0.433 mol) was added dropwise over 1hour, while the internal temperature was maintained between 0 and 15° C.After additional 0.5-1 hour, MeOH (35 mL, 0.87 mol) was added dropwiseover 0.5 hour and the batch was aged for additional 0.5 hour. Thereaction stream was then charged to a slurry of Pt/Al₂O₃ (5 wt %, 15 g)in THF (500 mL) at ambient temperature. The reaction mixture washydrogenated at 50-100 psig of H₂ at 20° C. for 18 hours, followed byadditional 6 hours at 50° C. Then, the catalyst was removed throughfiltration of a pad of SOLKA-FLOC® and washed with THF (400 mL). HCl(0.6 N, ˜1.2 L) was added dropwise to pH=2, maintaining the batchtemperature below 25° C. The batch was agitated at ambient temperaturefor 1 hour. Isopropyl acetate (300 mL) was added. The desired aqueousphase was separated and isopropyl acetate (400 mL) was added. Then, 5NNaOH (˜30 mL) was added dropwise to adjust pH=10. The organic phase wasseparated and treated with active carbon (Cuno active carbon, 10 g) atambient temperature for 1 hour. The carbon was removed throughfiltration and the filtrate was solvent switched to IPA at a volume of˜35 mL. The batch was warmed to 50° C. and water (30 mL) was addeddropwise. Then, the batch was seeded at 42° C. and additional water (50mL) was added dropwise over 2 hours. After addition, the batch wasgradually cooled to 10° C. and aged additional several hours beforefiltration. The wet cake was washed with 25% IPA in water (50 mL).Suction drying gave the Compound I-6 hemihydrate.

¹H NMR (d₆-DMSO) δ 7.27 (m, 4H), 7.17 (m, 1H), 6.81 (d, J=8.1, 2H), 6.45(d, J=8.1 Hz, 2H), 5.07 (s, br, 1H), 4.75 (s, 2H), 4.18 (d, J=7.0 Hz,1H), 3.05 (m, 2H), 2.47 (dd, J=13.0, 6.7 Hz, 1H), 2.40 (dd, J=13.0, 6.6Hz, 1H), 1.53 (m, 1H), 1.34 (m, 1H0, 1.22 (m, 2H).

¹³C NMR (d₆-DMSO) δ 146.5, 144.3, 129.2, 127.8, 127.4, 126.8, 126.7,114.0, 76.8, 64.4, 60.1, 42.1, 30.2, 27.2.

Step 6a. Preparation of Compound A-2

Step 6. Preparation of Compound I-7 from Compound I-6 and Compound A-2

To a three neck flask equipped with a N₂ inlet, a thermo couple probewas charged pyrrolidine hemihydrate I-6 (10.3 g), sodium salt A-2 (7.87g), followed by IPA (40 mL) and water (24 mL). 5 N HCl (14.9 mL) wasthen slowly added over a period of 20 minutes to adjust pH=3.3-3.5,maintaining the batch temperature below 35° C. Solid EDC hydrochloride(7.47 g) was charged in portions over 30 minutes. The reaction mixturewas aged at RT for additional 0.5-1 hour, aqueous ammonia (14%) wasadded dropwise to pH ˜8.6. The batch was seeded and aged for additional1 hour to form a slurry bed. The rest aqueous ammonia (14%, 53.2 mltotal) was added dropwise over 6 hours. The resulting thick slurry wasaged 2-3 hours before filtration. The wet-cake was displacement washedwith 30% IPA (30 mL), followed by 15% IPA (2×20 mL) and water (2×20 mL).The cake was suction dried under N₂ overnight to afford 14.3 g ofcompound I-7.

¹H NMR (DMSO) δ 10.40 (s, NH), 7.92 (d, J=6.8, 1H), 7.50 (m, 2H), 7.32(m, 2H), 7.29 (m, 2H), 7.21 (m, 1H), 7.16 (m, 2H), 6.24 (d, J=6.8, 1H),5.13 (dd, J=9.6, 3.1, 1H), 5.08 (br s, OH), 4.22 (d, J=7.2, 1H), 3.19(p, J=7.0, 1H), 3.16-3.01 (m, 3H), 2.65 (m, 1H), 2.59-2.49 (m, 2H), 2.45(br s, NH), 2.16 (ddt, J=13.0, 9.6, 3.1, 1H), 1.58 (m, 1H), 1.39 (m,1H), 1.31-1.24 (m, 2H).

¹³C NMR (DMSO) δ 167.52, 165.85, 159.83, 154.56, 144.19, 136.48, 135.66,129.16, 127.71, 126.78, 126.62, 119.07, 112.00, 76.71, 64.34, 61.05,59.60, 42.22, 31.26, 30.12, 27.09, 23.82.

The crystalline freebase anhydrous form I of Compound I-7 can becharacterized by XRPD by the following reflections with the d-spacing.

Position [°2 Theta] d-spacing [Å] 5.4897 16.09873 8.7494 10.1068311.0426 8.01261 16.4521 5.38818 16.6518 5.32402 17.4975 5.06857 18.8534.7071 21.2176 4.18756 21.7532 4.08564 22.7425 3.9101

EXAMPLE 2

Preparation of Compound I-7 from Compound I-6

To a three neck flask equipped with a N₂ inlet, a thermo couple probewas charged pyrrolidine hemihydrate I-6 (10.3 g), sodium salt A-2 (7.5g), followed by IPA (40 mL), pyridine (0.42 mL), and water (21 mL). 5 NHCl (14.9 mL) was then slowly added over a period of 20 minutes toadjust pH=3.1-3.7, maintaining the batch temperature below 25° C. Thebatch was cooled to 5-10° C. Solid EDC hydrochloride (7.5 g) was chargedin portions over 30 min to 1 hour, maintain the batch temperature below15° C. The reaction mixture was then aged between 5-10° C. foradditional 0.5-1 hour. IPA (8 mL) and water (7 mL) were added at rt.Aqueous ammonia (10%, ˜3.5 mL) was added dropwise to pH ˜7.9. The batchwas seeded and aged for additional 1 hour to form a slurry bed. Moreaqueous ammonia (10%, 32 mL) was added dropwise over 6-10 hours. Theresulting thick slurry was aged 2-3 h before filtration. The wet-cakewas displacement washed with 30% IPA (30 mL), followed by 15% IPA (2×20mL) and water (2×20 mL). The cake was suction dried under N₂ overnightto afford 14.3 g of compound I-7.

EXAMPLE 3

Immobilization of KRED Enzyme:

To a solution of sodium potassium phosphate (6.097 g, 3.5 mmol) in water(35 mL) at ambient temperature was added KRED enzyme (875 mg) and NADP(70 mg) and were dissolved with gentle agitation. Polymethacrylic resin,DIAION™ HP2MG, (MITSUBISHI CHEMICALS) (10 g) was added. The mixture wasgently agitated at 25° C. for at least 16 hours. The resin was drainedof all solution. The wet resin was washed with a solution of potassiumdibasic phosphate and subsequently drained. The wet resin was stored at4° C.

Ketone Reduction Procedure:

Immobilized KRED enzyme (120 mg) was added to a solution of ketone (50mg) dissolved in 90% IPA 10% water mixture containing immobilizedcarbonate (50 mg). The mixture was agitated at 25° C. for at least 24hours. The batch was filtered and concentrated.

EXAMPLE 4

Immobilization of KRED Enzyme with Resin Immobead™ 150A (IB-150A):

To a solution of 0.1M sodium potassium phosphate at pH 7.0 (20 mL) atambient temperature was added KRED enzyme with the SEQ. ID. NO. 1 (2 g)and dissolved with gentle agitation. Resin IB-150A (10 g) (commerciallyavailable from ChiralVision™, The Netherlands) was charged and aged at25° C. for 24-48 hours. The resin was filtered off all solution, andwashed three times with 0.1 M solution of sodium potassium phosphate atpH 7 and dried. The resin was stored at 4° C. for use.

Ketone Reduction to Preparation of Phenyl Alcohol by Immobilized KREDEnzyme:

To a 1 L flask was charged 360 ml IPA and 40 ml water, followed byketone I-3 (21 g, 73.1 mmol) and 1,4-diazabicyclooctane (41.0 g, 365mmol) at 25° C. The mixture was agitated until all was dissolved.Immobilized SEQ ID NO. 1 (10.5 g, 50 wt %) was charged to flask and thereaction mixture was heated to 50° C. with gentle agitation. Upon agingfor about 28 hours the reaction typically gave 98% conversion, 99% eeand 100:1 Dr. The reaction was cooled to 25° C. and filtered to removeimmobilized SEQ ID NO. 1 and rinsed with 100 ml (5 vol) IPA. Therecovered immobilized SEQ ID NO. 1 can be charged to fresh solution ofketone/DABCO IPA/water solution, and recycled up to 9 rounds whilereaching specifications for conversion and selectivity. After removingimmobilized enzyme the crude reaction solution was azeotropically driedto remove water and reduce reaction volume to ˜5 volumes IPA. Phosphoricacid (5 eq, 1:1 ratio with DABCO) was added at 25° C. and subsequentslurry was aged for 4 hours. The slurry was filtered to remove at least96% of DABCO salt from solution. Crude solution of phenyl alcohol wasdirectly used in subsequent step.

While the invention has been described and illustrated with reference tocertain particular embodiments thereof, those skilled in the art willappreciate that various changes, modifications and substitutions can bemade therein without departing from the spirit and scope of theinvention. It is intended, therefore, that the invention be defined bythe scope of the claims which follow and that such claims be interpretedas broadly as is reasonable.

SEQ ID NO. 1:Met Thr Asp Arg Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr1               5                   10                  15Gln Gly Ile Gly Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala            20                  25                  30Lys Val Val Ile Thr Gly Arg Arg Ala Asp Val Gly Glu Lys Ala Ala        35                  40                  45Lys Ser Ile Gly Gly Thr Asp Val Ile Arg Phe Val Gln His Asp Val    50                  55                  60Ser Asp Glu Ala Gly Trp Pro Lys Leu Phe Asp Thr Thr Glu Glu Ala65                  70                 75                   80Phe Gly Pro Val Thr Thr Val Val Asn Asn Ala Gly Ile Pro Met Val                85                  90                  95Lys Ser Val Glu Asp Thr Thr Thr Glu Glu Trp Arg Lys Leu Leu Ser            100                 105                 110Val Asn Leu Asp Gly Val Phe Phe Gly Ala Arg Leu Gly Ile Gln Arg        115                 120                 125Met Lys Asn Lys Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Val    130                 135                 140Phe Gly Ile Val Gly Asp Pro Thr Thr Gly Ala Tyr Cys Ala Ser Lys145                 150                 155                 160Gly Ala Val Arg Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu                165                 170                 175Lys Asp Tyr Asp Val Arg Val Asn Thr Val His Pro Gly Pro Ile Lys            180                 185                 190Thr Pro Met Met Asp Ser Tyr Glu Gly Ala Glu Glu Met Phe Ser Gln        195                 200                 205Arg Thr Lys Thr Pro Met Gly His Ile Gly Glu Pro Asn Asp Ile Ala    210                 215                 220Trp Val Cys Val Tyr Leu Ala Ser Asp Glu Ser Lys Phe Ala Thr Gly225                 230                 235                 240Ala Glu Phe Val Val Asp Gly Gly Phe Thr Ala Gln                245                 250

What is claimed is:
 1. A process of making compound I-6:

comprising: (a-2) reducing compound I-3:

in the presence of a ketoreductase (KRED) enzyme to produce compoundI-4:

(b-2) coupling compound I-4 with compound A-1, in the presence ofCatalyst D to produce compound I-5(a):

followed by deprotecting in situ with an acid to produce compound I-5(b)as a salt:

I-5(a), where R^(N)=P¹ or I-5 (b), where R^(N)=H I-5(a), where R^(N)=P¹or I-5(b) where, R^(N)=H; and (c-2) cyclizing and reducing compoundI-5(b) in the presence of Catalyst E to produce compound I-6 via I-6-1:

wherein P¹ is selected from the group consisting of Ac, Bn, Boc, Bz,Cbz, DMPM, FMOC, Ns, Moz, and Ts; and Y is selected from the groupconsisting of Cl, I, Br, and OTf; and R is selected from the groupconsisting of H, TMS, TES, TBDMS, TIPS and TBDPS; and R^(N) is P¹ or H.2. A process of making compound I-7:

comprising: (a-2) reducing compound I-3;

in the presence of a KRED enzyme to produce compound I-4:

(b-2) coupling compound I-4 with compound A-1, in the presence ofCatalyst D to produce compound I-5(a):

followed by deprotecting in situ with an acid to produce compound I-5(b)as a salt:

I-5(a), where R^(N)=P¹ or I-5 (b) where, R^(N)=H I-5(a), where R^(N)=P¹or I-5(b) where, R^(N)=H; (c-2) cyclizing and reducing compound I-5(b)in the presence of Catalyst E to produce compound I-6 via I-6-1:

and (d-2) coupling compound I-6 with compound A-2:

in the presence of a coupling agent and optionally including a base toproduce compound I-7;

wherein P¹ is selected from the group consisting of Ac, Bn, Boc, Bz,Cbz, DMPM, FMOC, Ns, Moz, and Ts; and Y is selected from the groupconsisting of Cl, I, Br, and OTf; and R is selected from the groupconsisting of H, TMS, TES, TBDMS, TIPS and TBDPS; and R^(N) is P¹ or H.3. A process of making compound I-6:

comprising: (a-1) reacting compound I-1:

in the presence of a solvent, an oxidizing agent, and Catalyst A to forman aldehyde in situ, followed by a condensation in the presence of NaCNor KCN and ammonium chloride and a protective reagent to producecompound I-2:

(b-1) reacting compound I-2 in the presence of a phenyl Grignard reagentto produce compound I-3:

(c-1) reducing compound I-3 in the presence of a KRED enzyme to producecompound I-4:

(d-1) coupling compound I-4 with compound A-1, in the presence ofCatalyst D to produce compound I-5(a):

followed by deprotecting in situ with an acid to produce compound I-5(b)as a salt:

I-5(a), where R^(N)=P¹ or I-5 (b) where, R^(N)=H I-5(a), where R^(N)=P¹or I-5(b) where, R^(N)=H; and (e-1) cyclizing and reducing compoundI-5(b) in the presence of Catalyst E to produce compound I-6 via I-6-1:

wherein P¹ is selected from the group consisting of Ac, Bn, Boc, Bz,Cbz, DMPM, FMOC, Ns, Moz, and Ts; and Y is selected from the groupconsisting of Cl, I, Br, and OTT; and R is selected from the groupconsisting of H, TMS, TES, TBDMS, TIPS and TBDPS; and R^(N) is P¹ or H.4. The process of claim 3, wherein in step (a-1): the solvent isselected from the group consisting of THF, MTBE, CH₂Cl₂, MeCN, EtOAc,i-PrOAc, Me-THF, hexane, heptane, DMAc, DMF, methyl cyclopentyl ether,toluene and combinations thereof; the oxidizing agent is selected fromthe group consisting of NaOCl, NaClO₂, PhI(OAc)₂, hydrogen peroxide;pyridine sulfur trioxide/Et₃N/DMSO and a Moffatt variant, PCC, DCC, aSwern oxidation or its variants, and TPAP/NMO; and Catalyst A is TEMPOor a TEMPO analogue.
 5. The process of claim 3, wherein P¹ in step (a-1)is Boc.
 6. The process of claim 3, wherein step (a-1) is carried out ata temperature of about 35° C.′ to about 45° C. in the presence of EtOAcor i-PrOAc.
 7. The process of claim 3, wherein in step (a-1) compoundI-2 can be prepared via a hydrogensulfite adduct.
 8. The process ofclaim 2, wherein the KRED enzyme in step (a-2) comprises the amino acidsequence set forth in SEQ NO.1 or an active fragment thereof.
 9. Theprocess of claim 2, wherein a cofactor recycling system is present instep (a-2).
 10. The process of claim 2, wherein the reaction in step(a-2) is carried out in a solvent selected from the group consisting of2-propanol, sec-butanol, iso-butanol, DMSO, DMF, DMAc, NMP, andcombinations thereof.
 11. The process of claim 2, wherein the reactionin step is carried out in a pH range of above about
 8. 12. The processof claim 2, wherein the reaction in step (a-2) is carried out at atemperature of about 30° C. to about 50° C.
 13. The process of claim 2,wherein Catalyst D used in the reaction in step (b-2) is selected fromthe group consisting of Pd(PPh₃)₄, PdCl₂, (PPh₃)₂PdCl₂, Pd(dppe)Cl,Pd(dppp)Cl₂, Pd(dppf)Cl₂, and Pd(OAc)₂/Ph₃P, in the presence or absenceof a catalytic amount of material selected from the group consisting ofCuI, CuBr, and CuCl.
 14. The process of claim 2, wherein the reaction instep (b-2) is carried out in the presence of a solvent selected from thegroup consisting of THF, IPA, MeOH, EtOH, n-PrOH, NMP, DMF, DMAc, MTBE,CH₂Cl₂, MeCN, Me-THF, methyl cyclopentyl ether, toluene, andcombinations thereof.
 15. The process of claim 2, wherein the reactionproduct in step (b-2) is isolated as a solid HCl salt.
 16. The processof claim 2, wherein compound I-5(b) salt reacts in step (c-2) with abase selected from the group consisting of Et₃N, i-Pr₂NEt, i-Pr₂NH,pyridine, lutidine, N-methyl morphine, t-BuOK, t-BuONa, t-BuOLi, NaH,NaHMDS, LiHMDS, and KHMDS to produce compound I-6-1 through anintramolecular cyclization reaction.
 17. The process of claim 2, whereincompound I-6-1 is reduced to compound I-6 in step (c-2) in the presenceof hydrogen gas and Catalyst E.
 18. The process of claim 3, wherein thereaction product in step (d-1) is isolated as a solid HCl salt.
 19. Aprocess of making compound I-6:

comprising: (c-2) cyclizing and reducing compound I-5(b) or a saltthereof:

in the presence of Catalyst E to produce compound I-6 via I-6-1:

wherein R is selected from the group consisting of H, TMS, TES, TBDMS,TIPS and TBDPS.
 20. The process of claim 19, further comprising: (d-2)coupling compound I-6 with compound A-2:

in the presence of a coupling agent and optionally including a base toproduce compound I-7: